Butyric acid is a non-toxic natural product found in butter in concentrations of up to approximately 5%. In the digestive system it is secreted as a product of microbial fermentation. In the colon it can reach mM concentrations.
It is known that butyric acid, whether in free form or more usually in the form of its alkali metal salts (hereinafter referred to as "butyric acid/salts"), displays antineoplastic activity. In particular, this activity is evidenced in the form of toxicity towards neoplastic cells, inhibition of cell proliferation, and induction of cytodifferentiation. Such activity has been demonstrated both in vitro and in vivo.
Thus, e.g. in a variety of tumor cells grown in vitro, there has been reported anti-tumor activity of butyric acid/salts due to the induction of morphological and biochemical changes. Some representative examples of affected cells derived from human sources are: neuroblastoma [Prasad and Kumar, Cancer 36:1338 (1975)]: leukemia [Collins et al. Proc. Natl. Acad. Sci. 75:2458 (1978)]; colon carcinoma [Dexter et al. Histochem. 16:137 (1984)] and Augeron and Laboisse, Cancer Res. 44:3961 (1984)]; pancreatic carcinoma [McIntyres et al, Euro. J. Cancer Clin. Onc. 20:265 (1984)]; kidney tumor cells [Heifetz et al. J. Biol. Chem. 256:6529 (1981)]; breast cancer [Stevens et al, Biochem. Biophys. Res. Comm. 119:132 (1984)]; prostatic carcinoma [Reese et al, Cancer Res. 45:2308 (1985)]; astrocytoma [McIntyre, J. Cell. Sci. 11:634 (1971)]; human epidermoid carcinoma [Marcher et al, Exp. Cell. Res. 117:95 (1978)]. Moreover, in all in vitro tests carried out by the present inventors, on leukemic cells isolated from myelogenous leukemic patients, butyric acid/salts was found to be the most potent cytotoxic and cytodifferentiating agent, being for example, more effective than retinoic acid, 1, 25-dihydroxy vitamin D and cytosine arabinoside.
Reported examples of in vivo application of butyric acid/salts are as follows. Patients with neuroblastoma received doses of up to 10 g./day, which produced no clinically detectable toxicity [Prasad, Life Sci. 27:1351 (1980)]. Treatment of a child with refractory acute myelogenous leukemia in relapse. with 0.5 g./kg./day, resulted in partial and temporary remission without detectable toxic effects [Novogrodsky et al. Cancer 51:9 (1983)]. Furthermore, the present inventors have treated a patient with acute myelogenous leukemia in relapse, with 1.0 g./kg./day for 10 days and 1.5 g./kg./day for an additional 6 days; the clinical follow up showed no adverse reaction [Rephaeli et al, Blood 68:192a (1986)]. Clinical trials with high dosages of butyric acid/salts resulted in no toxicity.
The selectivity of butyric acid/salts was demonstrated, in hitherto unpublished work (by M. Shaklai and E. Januszewiez) by inhibition of colony forming units, granulocytes and macrophages (CFU-GM), grown in soft agar, obtained from normal bone marrow and from peripheral blood of leukemic patients.
The main disadvantages of butyric acid/salts are a low intrinsic potency, a long induction period (4-5 days in vivo. 48 hours in vitro), a high clearance and rapid metabolization. Also, it is usually administered by infusion to peripheral veins, in high doses up to 1.5 g./kg./day, which procedure requires hospitalization, and is generally inconvenient for patients. Moreover, because of the high dosage, fluid overload and mild alkalosis may occur. Additionally, the unpleasant odor of butyric acid makes it socially unacceptable, and makes isolation of the patients desirable. Thus, although the antineoplastic activity of butyric acid/salts has been known for many years, the foregoing disadvantages have delayed or prevented its clinical application.
In order to overcome such disadvantages, the present invention provides derivatives of butyric acid which will be defined hereinbelow, and which have been found to inhibit proliferation, and to stimulate differentiation of malignant cells. These effects are unpredictably greater than those observed for butyric acid/salts. The derivatives provided by the invention can be utilized in greatly reduced amounts, compared with the parent acid or salts thereof. Thus, in the method of treatment, intermittent injection, or oral administration of the encapsulated drug (for example) can replace administration by continuous infusion. It should therefore be possible to reduce or prevent the need for hospitalization during the term of the patient's treatment. Because of the drug potency, fluid overload and alkalosis will be reduced or eliminated and the extent of odor unpleasantness will also be considerably reduced.
While the present invention is not to be regarded as restricted by any theory of action, nevertheless it is presently believed that the esters of the invention, because of their lipophilic character, are able to penetrate the lipoproteinic membranes of cells more efficiently than the relatively polar butyric acid or its salts, and that their lessened polar character causes a delay in their rate of metabolization, which results in further amplification of the physiological effects. It is moreover believed that under the influence of hydrolytic enzymes, the present derivatives undergo hydrolytic cleavage in vivo, releasing butyric acid and other non-toxic residues.
To the best of the inventors' belief, the compounds provided by the present invention are novel, with the exception of compounds having the formula Pr--C(.dbd.O)--O--CHR"--O--(O.dbd.)C--Pr, in which R" is hydrogen or methyl (see U.S. Pat. No. 4012526). These compounds are reported to promote animal growth when used as components of animal feeds, and to prevent fungal attack on such feeds, but do not appear to have been suggested for use as components of pharmaceutical compositions. The use of such compounds as antitumor agents is therefore also unknown.